Inbred SJL mice recapitulate human resistance to Cryptococcus infection due to differential immune activation.

IMPORTANCE: Cryptococcosis studies often utilize the common C57BL/6J mouse model. Unfortunately, infection in these mice fails to replicate the basic course of human disease, particularly hampering immunological studies. This work demonstrates that SJL/J mice can recapitulate human infection better than other mouse strains. The immunological response to Cryptococcus infection in SJL/J mice was markedly different from C57BL/6J and much more productive in combating this infection. Characterization of infected mice demonstrated strain-specific genetic linkage and differential regulation of multiple important immune-relevant genes in response to Cryptococcus infection. While our results validate many of the previously identified immunological features of cryptococcosis, we also demonstrate limitations from previous mouse models as they may be less translatable to human disease. We concluded that SJL/J mice more faithfully recapitulate human cryptococcosis serving as an exciting new animal model for immunological and genetic studies.

In utero complementation of a neural crest-derived melanocyte defect using cell directed gene transfer.

This study describes an in utero approach for overexpressing genes in a cell-type directed manner. It uses an avian leukosis retroviral expression system coupled with a transgenic mouse line expressing the viral receptor tv-a from a tissue-specific promoter (RCAS-TVA system) (Federspiel et al., 1994, and reviewed in Fisher et al., 1999). A transgenic mouse line was generated expressing tv-a from the Dopachrome tautomerase promoter (DCT-tv-a) in embryonic melanocyte precursors (melanoblasts). RCAS virus encoding beta-galactosidase (RCAS-LacZ) or tyrosinase (RCAS-Tyr) was injected in utero into embryonic day 12.5 albino (tyrosinase inactive) mouse embryos. Animals were analyzed for beta-galactosidase activity or tyrosinase activity (hair pigmentation). RCAS gene expression was detected in 44% and 25% of the transgenic mice, respectively. We demonstrate the RCAS-TVA system coupled with the DCT-tv-a line of mice can be used for in utero infection.

Generation of RCAS vectors useful for functional genomic analyses.

Avian leukosis type A virus-derived retroviral vectors have been used to introduce genes into cells expressing the corresponding avian receptor tv-a. This includes the use of Replication-Competent Avian sarcoma-leukosis virus (ASLV) long terminal repeat (LTR) with Splice acceptor (RCAS) vectors in the analysis of avian development, human and murine cell cultures, murine cell lineage studies and cancer biology. Previously, cloning of genes into this virus was difficult due to the large size of the vector and sparse cloning sites. To overcome some of the disadvantages of traditional cloning using the RCASBP-Y vector, we have modified the RCASBP-Y to incorporate "Gateway" site-specific recombination cloning of genes into the construct, either with or without HA epitope tags. We have found the repetitive "att" sequences, which are the targets for site-specific recombination, do not impair the production of infectious viral particles or the expression of the gene of interest. This is the first instance of site-specific recombination being used to generate retroviral gene constructs. These viral constructs will allow for the efficient transfer and expression of cDNAs needed for functional genomic analyses.

Neural crest-directed gene transfer demonstrates Wnt1 role in melanocyte expansion and differentiation during mouse development.

Wnt1 signaling has been implicated as one factor involved in neural crest-derived melanocyte (NC-M) development. Mice deficient for both Wnt1 and Wnt3a have a marked deficiency in trunk neural crest derivatives including NC-Ms. We have used cell lineage-directed gene targeting of Wnt signaling genes to examine the effects of Wnt signaling in mouse neural crest development. Gene expression was directed to cell lineages by infection with subgroup A avian leukosis virus vectors in lines of transgenic mice that express the retrovirus receptor tv-a. Transgenic mice with tva in either nestin-expressing neural precursor cells (line Ntva) or dopachrome tautomerase (DCT)-expressing melanoblasts (line DCTtva) were analyzed. We overstimulated Wnt signaling in two ways: directed gene transfer of Wnt1 to Ntva(+) cells and transfer of beta-catenin to DCTtva(+) NC-M precursor cells. In both methods, NC-M expansion and differentiation were effected. Significant increases were observed in the number of NC-Ms [melanin(+) and tyrosinase-related protein 1 (TYRP1)(+) cells], the differentiation of melanin(-) TYRP1(+) cells to melanin(+) TYRP1(+) NC-Ms, and the intensity of pigmentation per NC-M. These data are consistent with Wnt1 signaling being involved in both expansion and differentiation of migrating NC-Ms in the developing mouse embryo. The use of lineage-directed gene targeting will allow the dissection of signaling molecules involved in NC development and is adaptable to other mammalian developmental systems.

The use of expression profiling to study pigment cell biology and dysfunction.

Regulation of gene expression is a fundamental process by which cells respond to both intracellular and extracellular signals. For a pigment cell, alterations in gene expression regulate the processes of cell migration, lineage restriction, differentiation, type of pigment produced, and progression from a normal pigment cell to that of melanoma. To date, the identification of genes involved in normal pigment cell development has been accomplished by the cloning of individual mutant alleles, a single gene at a time. Current advances in technology have now made it possible to use expression profile analysis to investigate, on a genomic scale, the process of pigment cell development and function. This review compares and contrasts the methods of subtractive suppressive polymerase chain reaction (PCR) and differential display with that of cDNA microarray analysis.

cDNA microarrays detect activation of a myogenic transcription program by the PAX3-FKHR fusion oncogene.

Alveolar rhabdomyosarcoma is an aggressive pediatric cancer of striated muscle characterized in 60% of cases by a t(2;13)(q35;q14). This results in the fusion of PAX3, a developmental transcription factor required for limb myogenesis, with FKHR, a member of the forkhead family of transcription factors. The resultant PAX3-FKHR gene possesses transforming properties; however, the effects of this chimeric oncogene on gene expression are largely unknown. To investigate the actions of these transcription factors, both Pax3 and PAX3-FKHR were introduced into NIH 3T3 cells, and the resultant gene expression changes were analyzed with a murine cDNA microarray containing 2,225 elements. We found that PAX3-FKHR but not PAX3 activated a myogenic transcription program including the induction of transcription factors MyoD, Myogenin, Six1, and Slug as well as a battery of genes involved in several aspects of muscle function. Notable among this group were the growth factor gene Igf2 and its binding protein Igfbp5. Relevance of this model was suggested by verification that three of these genes (IGFBP5, HSIX1, and Slug) were also expressed in alveolar rhabdomyosarcoma cell lines. This study utilizes cDNA microarrays to elucidate the pattern of gene expression induced by an oncogenic transcription factor and demonstrates the profound myogenic properties of PAX3-FKHR in NIH 3T3 cells.

Isolation, genomic organization, and expression analysis of Men1, the murine homolog of the MEN1 gene.

The mouse homolog of the human MEN1 gene, which is defective in a dominant familial cancer syndrome, multiple endocrine neoplasia type 1 (MEN1), has been identified and characterized. The mouse Men1 transcript contains an open reading frame encoding a protein of 611 amino acids which has 97% identity and 98% similarity to human menin. Sequence of the entire Men1 gene (9.3 kb) was assembled, revealing 10 exons, with exon 1 being non-coding; a polymorphic tetranucleotide repeat was located in the 5'- flanking region. The exon-intron organization and the size of the coding exons 2-9 were well conserved between the human and mouse genes. Fluorescence in situ hybridization localized the Men1 gene to mouse Chromosome (Chr) 19, a region known to be syntenic to human Chr 11q13, the locus for the MEN1 gene. Northern analysis indicated two messages-2.7 kb and 3.1 kb-expressed in all stages of the embryo analyzed and in all eight adult tissues tested. The larger transcript differs from the smaller by the inclusion of an unspliced intron 1. Whole-mount in situ hybridization of 10.5-day and 11.5-day embryos showed ubiquitous expression of Men1 RNA. Western analysis with antibodies raised against a conserved C-terminal peptide identified an approximately 67-kDa protein in the lysates of adult mouse brain, kidney, liver, pancreas, and spleen tissues, consistent with the size of human menin. The levels of mouse menin do not appear to fluctuate during the cell cycle.

Cloning and tissue expression of the mouse ortholog of AIM1, a betagamma-crystallin superfamily member.

We report the isolation of the murine ortholog of AIM1, a human gene whose expression is associated with the reversal of tumorigenicity in an experimental model of melanoma. Mouse and human AIM1 are more than 90% identical in amino acid sequence in the betagamma-crystallin repeats and the C-terminal domain, and more than 75% identical in the extended N-terminal domain. Consistent with the isolated cDNA representing the authentic AIM1 ortholog, linkage analysis localized mouse Aim1 to proximal mouse Chromosome (Chr) 10 in a conserved linkage group with genes localized to human Chr band 6q21. Searches of EST databases identified a second AIM1-like gene in both mouse and human, suggesting the existence of a gene family. Northern analysis demonstrates Aim1 is expressed most abundantly in adult skin, lung, heart, liver, and kidney and is temporally regulated during embryogenesis. Aim1 is expressed highly in the shaft region of the hair follicles and the presumptive ectoderm, but not at detectable levels in melanocytes or melanocyte precursor cells.

Praja1, a novel gene encoding a RING-H2 motif in mouse development.

As part of a cloning strategy to identify genes involved in early mouse liver development we have isolated Praja1, a gene with similar sequences to the Drosophila melanogaster gene goliath (gl) which is involved in the fate of mesodermal cells ultimately forming gut musculatures, fat body, and the heart. Praja1 is a 2.1 kb gene encoding a putative 396 amino acid ORF and includes a COOH-terminal RING-H2 domain. Using the Jackson Laboratory BSS panel, we have localized Praja1 on chromosome X at 36 cM, which may be a candidate gene for mouse sla (sex linked sideroblastic anemia), near the X inactivation center gene, Xist. Northern blot analysis demonstrated three transcripts (3.1, 2.6 and 2.1 kb) in mRNA from adult mouse tissues brain, liver, and kidney as well as in mRNA from developing mouse embryos (days 7, 11, 15 and 17 post coitus, p.c.). In vitro transcription/translation yielded a product with an Mr of 59 kD. Immunohistochemical staining of in vitro liver explant cultures using a heterologous antibody against praja1 demonstrated cytoplasmic staining of cuboidal cells that have hepatocyte morphology and organization. The presence of the RING-H2 domain, a proline-rich region at the COOH-end, and regions rich in acidic amino acids, leads to the hypothesis that the Praja1 product is possibly involved in mediating protein-protein interactions, possibly as part of a protein sorting or transport pathway. This is strengthened by the similarity of Praja1 to rat Neurodap1, whose product has been shown to localize to the endoplasmic reticulum and golgi in brain.

Genomic organization of the murine Miller-Dieker/lissencephaly region: conservation of linkage with the human region.

Several human syndromes are associated with haploinsufficiency of chromosomal regions secondary to microdeletions. Isolated lissencephaly sequence (ILS), a human developmental disease characterized by a smooth cerebral surface (classical lissencephaly) and microscopic evidence of incomplete neuronal migration, is often associated with small deletions or translocations at chromosome 17p13.3. Miller-Dieker syndrome (MDS) is associated with larger deletions of 17p13.3 and consists of classical lissencephaly with additional phenotypes including facial abnormalities. We have isolated the murine homologs of three genes located inside and outside the MDS region: Lis1, Mnt/Rox, and 14-3-3 epsilon. These genes are all located on mouse chromosome 11B2, as determined by metaphase FISH, and the relative order and approximate gene distance was determined by interphase FISH analysis. The transcriptional orientation and intergenic distance of Lis1 and Mnt/Rox were ascertained by fragmentation analysis of a mouse yeast artificial chromosome containing both genes. To determine the distance and orientation of 14-3-3 epsilon with respect to Lis1 and Mnt/Rox, we introduced a super-rare cutter site (VDE) that is unique in the mouse genome into 14-3-3 epsilon by gene targeting. Using the introduced VDE site, the orientation of this gene was determined by pulsed field gel electrophoresis and Southern blot analysis. Our results demonstrate that the MDS region is conserved between human and mouse. This conservation of linkage suggests that the mouse can be used to model microdeletions that occur in ILS and MDS.

Melanocyte development in vivo and in neural crest cell cultures: crucial dependence on the Mitf basic-helix-loop-helix-zipper transcription factor.

The more than 20 different Mitf mutations in the mouse are all associated with deficiencies in neural crest-derived melanocytes that range from minor functional disturbances with some alleles to complete absence of mature melanocytes with others. In the trunk region of wild-type embryos, Mitf-expressing cells that coexpressed the melanoblast marker Dct and the tyrosine kinase receptor Kit were found in the dorsolateral neural crest migration pathway. In contrast, in embryos homozygous for an Mitf allele encoding a non-functional Mitf protein, Mitf-expressing cells were extremely rare, no Dct expression was ever found, and the number of Kit-expressing cells was much reduced. Wild-type neural crest cell cultures rapidly gave rise to cells that expressed Mitf and coexpressed Kit and Dct. With time in culture, Kit expression was increased, and pigmented, dendritic cells developed. Addition of the Kit ligand Mgf or endothelin 3 or a combination of these factors all rapidly increased the number of Dct-positive cells. Cultures from Mitf mutant embryos initially displayed Mitf-positive cells similar in numbers and Kit-expression as did wild-type cultures. However, Kit expression did not increase with time in culture and the mutant cells never responded to Mgf or endothelin 3, did not express Dct, and never showed pigment. In fact, even Mitf expression was rapidly lost. The results suggest that Mitf first plays a role in promoting the transition of precursor cells to melanoblasts and subsequently, by influencing Kit expression, melanoblast survival.

A high-resolution linkage map of the lethal spotting locus: a mouse model for Hirschsprung disease.

Mice homozygous for the lethal spotting (ls) mutation exhibit aganglionic megacolon and a white spotted coat owing to a lack of neural crest-derived enteric ganglia and melanocytes. The ls mutation disrupts the migration, differentiation, or survival of these neural crest lineages during mammalian development. A human congenital disorder, Hirschsprung disease (HSCR), is also characterized by aganglionic megacolon of the distal bowel and can be accompanied by hypopigmentation of the skin. HSCR has been attributed to multiple loci acting independently or in combination. The ls mouse serves as one animal model for HSCR, and the ls gene may represent one of the loci responsible for some cases of HSCR in humans. This study uses 753 N2 progeny from a combination of three intersubspecific backcrosses to define the molecular genetic linkage map of the ls region and to provide resources necessary for positional cloning. Similar to some cases of HSCR, the ls mutation acts semidominantly, its phenotypic effects dependent upon the presence of modifier genes segregating in the crosses. We have now localized the ls mutation to a 0.8-cM region between the D2Mit113 and D2Mit73/D2Mit174 loci. Three genes, endothelin-3 (Edn3), guanine nucleotide-binding protein alpha-stimulating polypeptide 1 (Gnas), and phosphoenolpyruvate carboxykinase (Pck1) were assessed as candidates for the ls mutation. Only Edn3 and Gnas did not recombine with the ls mutation. Mutational analysis of the Edn3 and Gnas genes will determine whether either gene is responsible for the neural crest deficiencies observed in ls/ls mice.

Modification and transfer into an embryonal carcinoma cell line of a 360-kilobase human-derived yeast artificial chromosome.

A neomycin resistance cassette was integrated into the human-derived insert of a 360-kilobase yeast artificial chromosome (YAC) by targeting homologous recombination to Alu repeat sequences. The modified YAC was transferred into an embryonal carcinoma cell line by using polyethylene glycol-mediated spheroplast fusion. A single copy of the human sequence was introduced intact and stably maintained in the absence of selection for over 40 generations. A substantial portion of the yeast genome was retained in hybrids in addition to the YAC. Hybrid cells containing the YAC retained the ability to differentiate when treated with retinoic acid. This approach provides a powerful tool for in vitro analysis because it can be used to modify any human DNA cloned as a YAC and to transfer large fragments of DNA intact into cultured mammalian cells, thereby facilitating functional studies of genes in the context of extensive flanking DNA sequences.